Mapping the Energy Landscape of Repeat Proteins using NMR-detected Hydrogen Exchange

Cortajarena, Aitziber L.; Mochrie, S. G. J.; Regan, Lynne

doi:10.1016/j.jmb.2008.02.046

Cited by 34 publications

(50 citation statements)

References 45 publications

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“…Such a description implies that thermodynamically, individual repeats unfold independently of each other and thus can populate partially folded configurations. This observation was confirmed when Cortajarena et al used equilibrium, NMR-detected hydrogen/deuterium exchange to observe sequential unfolding transitions for 2 CTPR proteins containing 2 and 3 repeats, respectively (20). However, we have also previously shown that kinetic folding of these 2 proteins at 20°C was 2-state over the limited denaturant concentration range measured (23).…”

Section: Biophysics and Computational Biologysupporting

confidence: 73%

“…Our analysis shows that the modular multistate equilibrium folding of the CTPRa proteins (20,23,38) is also observed in their kinetic folding and unfolding. Excitingly, our results are also completely consistent with the 2 computational studies on the folding CTPR proteins.…”

Section: Comparison Of Kinetics With Equilibrium and Computational Stmentioning

confidence: 71%

“…In particular, we and the Regan laboratory have used 2 series of designed consensus TPRs (a 34-aa, helix-turn-helix motif), called CTPR and CTPRa proteins, to investigate the dependence of thermodynamic characteristics and denatured state on increasing repeat number ( Fig. 1; the 2 series only differ by a 2-aa substitution per repeat) (20)(21)(22)(23). These studies have highlighted the 1-dimensionality and changing equilibrium properties of increasing repeat number by showing that the thermodynamic unfolding transition can be described and, importantly, predicted by an Ising model that uses nearest-neighbor interactions within a 1-dimensional lattice (22).…”

Section: Biophysics and Computational Biologymentioning

confidence: 99%

See 2 more Smart Citations

Exploring the folding energy landscape of a series of designed consensus tetratricopeptide repeat proteins

Javadi

Main

2009

Proc. Natl. Acad. Sci. U.S.A.

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Repeat proteins contain short, tandem arrays of simple structural motifs (20−40 aa). These stack together to form nonglobular structures that are stabilized by short-range interactions from residues close in primary sequence. Unlike globular proteins, they have few, if any, long-range nonlocal stabilizing interactions. One ubiquitous repeat is the tetratricopeptide motif (TPR), a 34-aa helix-turn-helix motif. In this article we describe the folding kinetics of a series of 7 designed TPR proteins that are assembled from arraying identical designed consensus repeats (CTPRa n ). These range from the smallest 2-repeat protein to a large 10-repeat protein (≈350 aa). In particular, we describe how the energy landscape changes with the addition of repeat units. The data reveal that although the CTPRa proteins have low local frustration, their highly symmetric, modular native structure is reflected in their multistate kinetics of unfolding and folding. Moreover, although the initial folding of all CTPRa n proteins involves a nucleus with similar solvent accessibility, their subsequent folding to the native structure depends directly on repeat number. This corresponds to an increasingly complex landscape that culminates in CTPRa10 populating a misfolded, off-pathway intermediate. These results extend our current understanding of the malleable folding pathways of repeat proteins and highlight the consequences of adding identical repeats to the energy landscape.

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Section: Biophysics and Computational Biologysupporting

confidence: 73%

Section: Comparison Of Kinetics With Equilibrium and Computational Stmentioning

confidence: 71%

Section: Biophysics and Computational Biologymentioning

confidence: 99%

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Exploring the folding energy landscape of a series of designed consensus tetratricopeptide repeat proteins

Javadi

Main

2009

Proc. Natl. Acad. Sci. U.S.A.

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“…Thus, although it has been shown that at equilibrium CTPRan and CTPRn proteins have the propensity for multistate folding and the population of stable intermediates, 23,24,36 our data show that this observable multistate folding can be modulated and is determined by the total stability of the protein and, consequently, the stability of the remaining partially folded species under the unfolding conditions. If they are unstable the protein acts as an observably two-state unfolding system (e.g., CTPRa2 and CTPRa3), with intermediates being high-energy and not populated.…”

Section: Discussioncontrasting

confidence: 55%

“…11,12,17 NMR and equilibrium chemical denaturation studies on the CTPR2 and CTPR3 proteins suggest that, despite the apparent cooperative equilibrium unfolding, intermediate states with frayed terminal helices are populated through the denaturation transition. 23,24 Further, Regan and coworkers have used DSC to show that all the CTPRa proteins undergo multistate equilibrium folding under their conditions. 20 This multistate folding was elegantly described by fitting and modeling the thermodynamic unfolding transition of chemical denaturations to these repeat proteins using a homozipper Ising model.…”

Section: Introductionmentioning

confidence: 99%

Modulation of the multistate folding of designed TPR proteins through intrinsic and extrinsic factors

et al. 2012

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Tetratricopeptide repeats (TPRs) are a class of all alpha-helical repeat proteins that are comprised of 34-aa helix-turn-helix motifs. These stack together to form nonglobular structures that are stabilized by short-range interactions from residues close in primary sequence. Unlike globular proteins, they have few, if any, long-range nonlocal stabilizing interactions. Several studies on designed TPR proteins have shown that this modular structure is reflected in their folding, that is, modular multistate folding is observed as opposed to two-state folding. Here we show that TPR multistate folding can be suppressed to approximate two-state folding through modulation of intrinsic stability or extrinsic environmental variables. This modulation was investigated by comparing the thermodynamic unfolding under differing buffer regimes of two distinct series of consensus-designed TPR proteins, which possess different intrinsic stabilities. A total of nine proteins of differing sizes and differing consensus TPR motifs were each thermally and chemically denatured and their unfolding monitored using differential scanning calorimetry (DSC) and CD/fluorescence, respectively. Analyses of both the DSC and chemical denaturation data show that reducing the total stability of each protein and repeat units leads to observable two-state unfolding. These data highlight the intimate link between global and intrinsic repeat stability that governs whether folding proceeds by an observably two-state mechanism, or whether partial unfolding yields stable intermediate structures which retain sufficient stability to be populated at equilibrium.

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Structural and dynamic characterization of a freestanding acyl carrier protein involved in the biosynthesis of cyclic lipopeptide antibiotics

et al. 2017

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Friulimicin is a cyclic lipodecapeptide antibiotic that is produced by Actinoplanes friuliensis. Similar to the related lipopeptide drug daptomycin, the peptide skeleton of friulimicin is synthesized by a large multienzyme nonribosomal peptide synthetase (NRPS) system. The LipD protein plays a major role in the acylation reaction of friulimicin. The attachment of the fatty acid group promotes its antibiotic activity. Phylogenetic analysis reveals that LipD is most closely related to other freestanding acyl carrier proteins (ACPs), for which the genes are located near to NRPS gene clusters. Here, we report that the solution NMR structure of apo-LipD is very similar to other four-helix bundle forming ACPs from fatty acid synthase (FAS), polyketide synthase, and NRPS systems. By recording NMR dynamics data, we found that the backbone motions in holo-LipD are more restricted than in apo-LipD due to the attachment of phosphopantetheine moiety. This enhanced stability of holo-LipD was also observed in differential scanning calorimetry experiments. Furthermore, we demonstrate that, unlike several other ACPs, the folding of LipD does not depend on the presence of divalent cations, although the presence of Mg 21 or Ca 21 can increase the protein stability. We propose that small structural rearrangements in the tertiary structure of holo-LipD which lead to the enhanced stability are important for the cognate enzyme recognition for the acylation reaction. Our results also highlight the different surface charges of LipD and FAS-ACP from A. friuliensis that would allow the acyl-CoA ligase to interact preferentially with the LipD instead of binding to the FAS-ACP.

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Mapping the Energy Landscape of Repeat Proteins using NMR-detected Hydrogen Exchange

Cited by 34 publications

References 45 publications

Exploring the folding energy landscape of a series of designed consensus tetratricopeptide repeat proteins

Exploring the folding energy landscape of a series of designed consensus tetratricopeptide repeat proteins

Modulation of the multistate folding of designed TPR proteins through intrinsic and extrinsic factors

Structural and dynamic characterization of a freestanding acyl carrier protein involved in the biosynthesis of cyclic lipopeptide antibiotics

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